verbatim): The ryanodine receptor (RyR)/calcium (Ca) release channel (CRC) is required for excitation-contraction (EC) coupling in cardiac and skeletal muscle. The long term objective of this project is to elucidate structure-function relationships of the skeletal (RyR1) and cardiac (RyR2) CRCs. In the past 5 years important advances have been achieved in terms of identifying functional domains of the channels and in understanding the role of the FK506 binding protein (FKBP12/12.6) in regulating RyR function. Significant new findings and preliminary data include: 1) FKBP12/12.6 stabilize RyR1 and RyR2 function; 2) identification of the binding site for FKBP12/12.6 on RyR1 and RyR2; 2) RyRs are physically bound to neighboring RyRs and 2 or more channels can exhibit coupled gating in planar lipid bilayers (believed to be important for controlling EC coupling); 3) identification of the macromolecular signaling complex comprised of RyR, FKBP12/12.6, and protein kinase A (PKA), the phosphatases PP1 and PP2A and their corresponding anchoring/adaptor proteins; 4) PKA phosphorylation of RyR2 dissociates FKBP12.6 and regulates channel function; and 5) RyR2 function is defective in failing hearts due to PKA hyperphosphorylation of the channel which dissociates FKBP12.6 resulting in """"""""leaky"""""""" channels.
The aims of this proposal are designed to extend these observations in new directions to expand our understanding of the mechanisms that regulate RyR channel function and their role in human diseases.
Aim 1 proposes to use site-directed mutagenesis and single channel recordings in planar lipid bilayers to identify key amino acid residues that form the FKBP12/12.6 binding sites in RyR1 and RyR2.
Aim 2 is to use yeast two-hybrid screens and GST-pull downs to determine the components of the RyR1 and RyR2 macromolecular signaling complexes that regulate FKBP12/12.6 binding to the channels.
Aim 3 proposes to identify regions of RyRl and RyR2 required for tetramer formation, and for binding between channels. Each of the molecules in the RyR macromolecular complex are potentially novel targets for probing mechanisms underlying EC coupling function and for treating human diseases. RyR2 regulation is defective in human heart failure and RyR1 is genetically linked to human diseases including malignant hyperthermia and central core disease. Pharmacologic manipulation, targeted at the regulation of FKBP12/12.6 binding to the channels and at phosphorylation/dephosphorylation of the channels, could ultimately provide novel approaches to treating these and other diseases.
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